Association between hemoglobin A1c, Vitamin C, and microbiome in diabetic foot ulcers and intact skin: A cross‐sectional study

Abstract Background and Aims Diabetic foot ulcers (DFUs) add billions of dollars to the direct annual costs associated with diabetes. Despite various treatments, many DFUs do not heal and become infected. Both skin‐associated microbial communities and glycemic control are believed to be important in nonhealing DFUs. Recent studies have linked serum Vitamin C levels with glycemic control and DFUs. This cross‐sectional study assessed skin microbiome in DFUs, intact diabetic skin, and nondiabetic skin to identify correlations between hemoglobin A1c (HbA1c), Vitamin C, and microbial community structure. Correlations between Vitamin C, HbA1c, wound size, and ulcer duration were also determined. Methods Participants had their DFUs or intact skin culture swabbed. HbA1c was obtained via point‐of‐care fingerstick testing and serum Vitamin C was obtained via venipuncture. All participants completed a dietary questionnaire. Participants with ulcers were stratified into the controlled (≤8.0%) or uncontrolled (>8.0%) HbA1c group. Analysis of microbial communities was performed via 16S ribosomal RNA (rRNA) gene amplicon sequencing and bacterial load was measured by the domain‐level quantitative polymerase chain reaction of the 16S rRNA gene. Results Forty‐two patients were recruited over 6 months. Bacteria from the genera Staphylococcus and Stenotrophomonas were present in all samples and often dominant, but a shift towards anaerobic pathogenic taxa was observed in ulcers. No global significant differences were observed for HbA1c and Vitamin C levels in the microbial community structure (R < 0.013/p > 0.375). Bacterial loads were 4–5 orders of magnitude higher in ulcers than in intact skin samples. Bacterial load was not significantly higher in the uncontrolled HbA1c group (p = 0.67). Larger wound sizes (p = 0.46) were observed in the uncontrolled HbA1c group compared to the control. Lower Vitamin C levels (p = 0.002) were observed in the uncontrolled HbA1c group compared to nondiabetic controls. Conclusion Understanding the link between Vitamin C and HbA1c and DFU microbiome may aid in new therapies.


| INTRODUCTION
Diabetic foot ulcers (DFUs) add 9 to 13 billion dollars to direct annual costs associated with diabetes. 1,2 Despite treatment, a high proportion of DFUs become infected, resulting in some form of lower extremity amputation (LEA). 3 In addition to increased microbial load and pathogenic shift of DFU-associated microbiomes, clinical factors have been associated with slow healing DFUs, including poor glycemic control 4 and Vitamin C deficiency. 5,6 According to Lane et al., 7 hemoglobin A1c (HbA1c) levels > 8% are associated with increased likelihood of LEA among patients with DFUs. Hyperglycemia-induced oxidative stress promotes harmful biofilm formation and can lower overall microbial diversity in DFUs. 8,9 Conversely, antioxidants like Vitamin C have been found to prevent biofilm formation in vitro, 10 suggesting that Vitamin C may influence the microbiome in DFUs. In addition, recent studies have noted a protective role of Vitamin C in glycemic control, 11,12 and thus, Vitamin C supplementation is being utilized as a part of diabetes management. 13 Recent studies have demonstrated that microbial community structure and clinical factors can independently impair the healing of DFUs, but few studies have examined associations between these factors. Pang et al. 14 measured significantly different microbial αdiversity on intact skin in patient groups with a differing duration of diabetes using high-throughput 16S ribosomal RNA (rRNA) sequencing. Their findings showed dynamic changes in intact skin microbiome during the progression of diabetes, suggesting that the duration of diabetes influences skin microbiota. However, they did not evaluate other, more objective clinical factors that may influence skin microbiota. Gardner et al. 15 evaluated the association between microbial community structure, HbA1c levels in DFUs, and several other clinical factors, but did not compare microbial community structure in DFUs with that of intact diabetic skin (IDS) and intact nondiabetic skin (NDS). They found that poor glycemic control was associated with ulcers containing a high relative abundance of Staphylococcus and Streptococcus.
In this cross-sectional study, we evaluated associations between HbA1c, Vitamin C, and microbial community structure in DFUs, IDS, and NDS. Cultivation-independent molecular techniques were used to interrogate microbial communities, including bacterial load measurements using quantitative PCR (qPCR), and microbial community structure analysis using 16S rRNA gene amplicon sequencing.   16 In patients without diabetes or ulcers, intact skin of bilateral feet was swabbed at the equivalent location of the gender-and age-matched patient with DFU. Samples were immediately frozen in collection tubes at −2°C to −5°C and sent to the Genome Research Core (GRC) at the University of Illinois at Chicago (UIC), Chicago, IL for processing.

| RESEARCH DESIGN AND METHODS
HbA1c was obtained via a point-of-care fingerstick (A1CNow+; PTS Diagnostics) by K. P. S. T. or J. O. If HbA1c had already been obtained within the past 3 months, finger-sticking was deferred, and the value in the medical record was used. Approximately 4 cc of serum was collected via venipuncture from the antecubital fossa using previously described techniques 17,18 for Vitamin C testing and centrifuged in-house by K. P. S. T. according to the Quest Diagnostics protocol.
The serum component was transferred into a separate clean, plastic, and light-protected transport tube and frozen before pick-up within 48 h by Quest Diagnostics.
In addition, self-reported fruit and vegetable (FV) intake was assessed using a 12-question dietary assessment survey (Appendix A).
Patients were asked to report the frequency of consumption of fruits and vegetables on a nine-point scale ranging from "never" to "2 or more times per day."

| Patient stratification
Patients with diabetes were stratified into two groups: controlled  19 Primers, probes, and double-stranded synthetic DNA standards (gBLOCKs) were synthesized by Integrated DNA Technologies. Analysis was performed using a ViiA7 Real-Time PCR instrument (Thermo Fisher Scientific) and with an 8-order of magnitude standard dilution series for absolute quantification. Genomic DNA was also used as a template for amplification of microbial 16S rRNA gene amplicons using a two-stage PCR protocol as described previously. 20 The primer set 515F modified and 806R modified, 21  Forward and reverse reads were merged using the software package PEAR. 24 Merged reads were trimmed to remove ambiguous nucleotides, primer sequences, and trimmed based on the quality threshold of p = 0.01. Merged reads that lacked either primer sequences or reads that were shorter than 225 bases were discarded.
Chimeric sequences were identified and removed using the USEARCH algorithm with comparison to the SILVA v132 reference sequence database. 25,26 Amplicon sequence variants (ASVs) were identified using DADA2. 27 The representative sequences for each ASVs were annotated using the Naïve Bayesian classifier included in DADA2 with the SILVA v132 reference sequence database. 28 Basic annotation pipelines were performed by the Research Informatics Core at the UIC.

| Outcomes
Primary outcome measures were associations between the microbiome (load, diversity, and pathogenic shift), HbA1c, and Vitamin C levels in DFU, IDS, and NDS samples. Secondary outcome measures examined the association between HbA1c, Vitamin C, wound size, and ulcer chronicity. For patient characteristics, wound characteristics, and the dietary assessment, differences were assessed using ANOVA, Fisher's exact test, Mann-Whitney U test, χ 2 test, and multiple linear regression analysis.

| RESULTS
The study included a total of 42 patients: 25 patients with DFU (7 patients with cHbA1c and 18 patients with uHbA1c), and 17 genderand age-matched controls (±4 years) without diabetes or ulcers. The characteristics of the study population are summarized in Table 1.
The prevalence of Vitamin C deficiency in the DFU sample was 41.8% and 0% in the control group without diabetes or ulcer. While Vitamin C levels were comparable between those with wellcontrolled diabetes and nondiabetic controls, significantly lower Vitamin C levels were observed in the uHbA1c group compared to nondiabetic controls (p = 0.002; Table 2). Larger wound sizes were noted in the uHbA1c group compared to the cHbA1c group (p = 0.46; Table 2). Mean ulcer duration at the time of enrollment was higher in the uHbA1c group (p = 0.28; Table 2 Table 6). The overall regression was not statistically significant, F(6,59) = 0.818, p = 0.541.
The overall microbial community structure in DFU samples was distinct from that observed in IDS and NDS samples (Figure 2A,B).
Significant differences were observed between the three sample groups (one-way ANOSIM global R = 0.383/p = 0.001; Figure 2A).
However, no global significant differences were observed for HbA1c relative abundances of Stenotrophomonas and Staphylococcus and higher relative abundance of putative pathogens relative to NDS and IDS samples (Figure 4). Streptococcus, and Enterococcus, some of which have been shown to being pathogenic or opportunistic pathogens. 35 Actinomyces are anaerobic, Gram-positive bacteria that have been implicated in endodontic infections. 36 Anaerococcus are another anaerobic, Grampositive bacteria commonly associated with gastrointestinal and urogenital disorders. 37 Finegoldia are also anaerobic, Gram-positive bacteria that induce inflammation by activating neutrophils. 38 Streptococcus are aerobic, Gram-positive bacteria that are commensal and pathogenic. They are associated with a number of disorders, including wound and skin infections, dental caries, and pneumonia. 39 A recent study by Eydou et al. 40 found that Vitamin C is associated with decreased Streptococcus growth and biofilm formation in specimens obtained from dental caries. Furthermore, it was found that the effects of Vitamin C were superior to gentamicin. 41 Finally, Enterococcus are facultatively anaerobic, Gram-positive bacteria that serve as a major causative agent of infections due to their multidrug resistance. 41 Anaerobic bacteria are prevalent in DFU, but many may go undetected with standard culture swabs. 42 We note, however, that not all members of these bacterial genera are pathogens, and that future studies employing long-read amplicon sequencing and shotgun metagenome sequencing will be needed to fully evaluate the taxonomy and functional capabilities of these organisms in the wound environment.

| Vitamin C dietary assessment survey
Infected, nonhealing DFUs possess a mixed microbial community characterized by elevated microbial load, decreased microbial diversity, and community shift towards putatively pathogenic taxa. 43 Historically, cultivation-dependent analyses of DFU microbial community structure favored invasive tissue biopsies over swabs. 44 However, cultivation approaches will also be limited by a priori knowledge of ideal growth conditions for the polymicrobial community and may miss substantial microbial diversity. Cultivation-independent approaches are essential for capturing simultaneously aerobic/microaerophilic/ anaerobic microorganisms present in the wound environment.
Cultivation-independent molecular tools have been recently used for the characterization of DFU-associated microbial community structure.
Travis et al. 35 observed that wound swabs could effectively be used for DFU microbial community analyses when coupled with genomic DNA extraction followed by high-throughput sequencing of microbial 16S rRNA gene amplicons. Such an approach has multiple advantages, due to less invasive sampling and a broader target range of known and potential pathogens by avoiding the need for cultivation. In addition, qPCR can be employed to quantify microbial load using assays targeting microbial rRNA genes. As a result, cultivation-independent analyses based on 16S rRNA gene amplicon sequencing are replacing cultivation-based swab analyses. 45 Future approaches may also utilize shotgun metagenome sequencing to avoid targeted amplification of single genes, thereby providing more functional information including the presence or absence of pathogenicity genes and antibiotic resistance genes. Transcriptional profiling may also be employed to identify expressed genes of pathogens within the wound environment.
In this study, metric multidimensional scaling plots showed significantly different bacterial communities between the skin types, and the difference was greater with DFU. We further stratified DFU patients into controlled and uncontrolled HbA1c groups to determine if a relationship between glycemic control and bacterial characteristics exists. We observed that while DFU bacterial loads were lower in the cHbA1c group relative to those in the uHbA1c group, the difference was not significant. However, HbA1c was significantly predictive of bacterial load when controlling for Finally, previous studies have found differences in skin microbiota in males compared to females. 49 For instance, Jnana et al. 49 found an increase in facultative anaerobes such as Proteus and Burkholderia in males compared to females. Gender differences could not sufficiently be accounted for in this study due to the small female cohort (7/42). Therefore, gender differences may be a potential confounding factor in this study. Furthermore, while there is a potential technical limitation due to swabbing, it is less invasive than collecting a tissue specimen and therefore commonly used in microbial analysis studies. 15